Molecular reaction mechanisms of heterotrimeric G-Proteins

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Heterotrimeric G-proteins are central switches within signalling pathways in cells. These are usually switched on via G-protein coupled receptors (GPCR), which catalyse the exchange from GDP to GTP. This causes the breakup of the heterotrimeric complex into its subunits and activates important signalling pathways. They are switched off again by GTP hydrolysis within the Galpha-subunit. GPCRs are currently investigated by numerous research groups worldwide, especially due to their importance in pharmacology. In contrast, we will investigate the less detailed studied hydrolysis of Galpha-proteins by a broad, integrative approach using time-resolved FTIR difference-spectroscopy and biomolecular simulations. The Galpha-subunits consist of the G-domain, conserved also in small GTPases, and an additional all-alpha domain. Several protein structures of Galpha obtained from X-ray crystallography are already available. However, non-hydrolysable GTP analogues were used, which interfere seriously with the catalytic centre. Complementary, time-resolved FTIR-spectroscopy can resolve the dynamics of the protein under physiological conditions and its interaction with atomic detail using the natural nucleotide. We have applied this approach already successfully for many small GTPases. In our preliminary work, we were able to transfer this approach also to Galpha-proteins. In order to decode detailed molecular information from the IR spectra, QM/MM calculations are applied in addition. Here we want to elucidate the role of the catalytically important amino acids. Besides a glutamine, stabilizing the nucleophilic water, in particular the role of the catalytic arginine will be determined. The latter is, in contrast to small GTPases, an intrinsic residue within the catalytic centre. Further, we want to understand how RGS-proteins, unlike GAPs of small GTPases, are able to further accelerate hydrolysis, without direct interaction with the nucleotide. This regulates the interruption of signalling pathways. Besides wildtype protein, we want to determine especially dysfunctions of the protein induced by mutations of the arginine and the glutamine. These mutations play an important role in severe diseases as the McCune-Albright syndrome and cancer. We will start our investigations with the inhibitory Gi and the activating Gs. Later it is planned to investigate Gq and Gt, to elucidate mechanistic alterations and similarities. Further diseases like cholera and pertussis are mediated by toxins, modifying Galpha-proteins by ADP-ribosylation. The mechanism of this modification will be investigated in detail.